The critical immunological feature of visceral leishmaniasis is the complete absence of cell-mediated immunity to leishmanial antigens. Patients have been shown to have negative intradermal skin tests to Leishmanin, absent lymphocyte blastogenesis, and decreased IL-2 and IFN-gamma production in response to parasite antigens. Experimental models of visceral leishmaniasis suggest that the disease is characterized not by the lack of cytokine production per se, but by the production of potentially counterprotective cytokines. We sought to maximize assay sensitivity by using semiquantitative RT-PCR techniques to analyze cytokine mRNA extracted from lesional tissue (bone marrow and spleen). In preliminary data we provided evidence that IL-10 mRNA is present at relatively high levels in active kala- azar, being at or below the limits of detectability after treatment, and absent in uninfected controls. The cellular source of this potentially downregulatory cytokine is being investigated in patients from India by repeating the above experiments using bone marrow cells positively and negatively selected for B cells, T cells, or monocytes. In addition, we now have available for analysis splenic tissue from Indian kala-azar patients during the course of their therapy with antimony alone, or with antimony plus IFN-gamma. The ability of Leishmania to evade the microbicidal activities of their host macrophages is obviously key to their successful parasitism. The manner by which they accomplish this is being investigated in vitro by studying the effect of infection on the production of proinflammatory cytokines involved in macrophage activation and intracellular killing. The parasites alone failed to trigger expression of any cytokine message. More importantly, costimulation of the macrophages with parasites and potent activators, LPS or killed mycobacteria, led to a striking downregulation of IL-1, TNF-alpha, NO synthase, and particularly IL-12. In contrast, IL-10, which is known to downregulate disease controlling TH1 responses, was either not affected or in some cases upregulated by the parasite. These results are consistent with the ability of the parasite to establish itself intracellularly during the early stages of infection and to persist by evading host immune responses.